Irreversible electroporation to create tissue scaffolds

ABSTRACT

The present invention provides engineered tissue scaffolds, engineered tissues, and methods of using them. The scaffolds and tissues are derived from natural tissues and are created using non-thermal irreversible electroporation (IRE). Use of IRE allows for ablation of cells of the tissue to be treated, but allows vascular and neural structures to remain essentially unharmed. Use of IRE thus permits preparation of thick tissue scaffolds and tissues due to the presence of vasculature within the scaffolds. The engineered tissues can be used in methods of treating subjects, such as those in need of tissue replacement or augmentation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. patent application Ser.No. 12/432,295 filed Apr. 29, 2009, which published as U.S. PatentApplication Publication No. 2009/0269317 on Oct. 29, 2009, and whichrelies on the disclosure of and claims the benefit of the filing date ofU.S. Provisional Patent Application No. 61/125,840, filed Apr. 29, 2008.The entire disclosures of each of these applications are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of biomedical engineering.More specifically, the invention relates to methods of producingnaturally-derived scaffolds for creation of tissues for medical uses,and tissues created from those scaffolds.

Description of Related Art

Tissue engineering holds great promise for treating some of the mostdevastating diseases of our time. Because engineered tissue and organreplacements can be developed in a laboratory, therapies can potentiallybe delivered on a large scale, for multiple disease states with dramaticreduction in waiting times for patients. The concept of engineeringtissue using selective cell transplantation has been appliedexperimentally and clinically for a variety of disorders, including thesuccessful use of engineered bladder tissue for bladder reconstruction,engineered injectable chondrocytes for the treatment of vesicoureteralreflux and urinary incontinence, and vascular grafts.

For clinical use for humans, the process involves the in vitro seedingand attachment of human cells onto a scaffold. Once seeded, the cellsproliferate, migrate into the scaffold, and differentiate into theappropriate cell type for the specific tissue of interest whilesecreting the extracellular matrix components required to create thetissue. The three dimensional structure of the scaffold, and inparticular the size of pores and density of the scaffold, is importantin successful proliferation and migration of seeded cells to create thetissue of interest. Therefore, the choice of scaffold is crucial toenable the cells to behave in the required manner to produce tissues andorgans of the desired shape and size.

To date, scaffolding for tissue engineering has usually consisted ofnatural and synthetic polymers. Methods known in the art for formingscaffolds for tissue engineering from polymers include solvent-casting,particulate-leaching, gas foaming of polymers, phase separation, andsolution casting. Electrospinning is another popular method for creatingscaffolds for engineered tissues and organs, but widely used techniquessuffer from fundamental manufacturing limitations that have, to date,prevented its clinical translation. These limitations result from thedistinct lack of processes capable of creating electrospun structures onthe nano-, micro-, and millimeter scales that adequately promote cellgrowth and function.

Of fundamental importance to the survival of most engineered tissuescaffolds is gas and nutrient exchange. In nature, this is accomplishedby virtue of microcirculation, which is the feeding of oxygen andnutrients to tissues and removing waste at the capillary level. However,gas exchange in most engineered tissue scaffolds is typicallyaccomplished passively by diffusion (generally over distances less than1 mm), or actively by elution of oxygen from specific types of materialfibers. Microcirculation is difficult to engineer, particularly becausethe cross-sectional dimension of a capillary is only about 5 to 10micrometers (μm; microns) in diameter. As yet, the manufacturingprocesses for engineering tissue scaffolds have not been developed andare not capable of creating a network of blood vessels. Currently, thereare no known tissue engineering scaffolds with a circulation designedinto the structure for gas exchange. As a result, the scaffolds fortissues and organs are limited in size and shape.

In addition to gas exchange, engineered tissue scaffolds must exhibitmechanical properties comparable to the native tissues that they areintended to replace. This is true because the cells that populate nativetissues sense physiologic strains, which can help to control tissuegrowth and function. Most natural hard tissues and soft tissues areelastic or viscoelastic and can, under normal operating conditions,reversibly recover the strains to which they are subjected. Accordingly,engineered tissue constructs possessing the same mechanical propertiesas the mature extracellular matrix of the native tissue are desirable atthe time of implantation into the host, especially load bearingstructures like bone, cartilage, or blood vessels.

There are numerous physical, chemical, and enzymatic ways known in theart for preparing scaffolds from natural tissues. Among the most commonphysical methods for preparing scaffolds are snap freezing, mechanicalforce (e.g., direct pressure), and mechanical agitation (e.g.,sonication). Among the most common chemical methods for preparingscaffolds are alkaline or base treatment, use of non-ionic, ionic, orzwitterionic detergents, use of hypo- or hypertonic solutions, and useof chelating agents. Among the most common enzymatic methods forpreparing scaffolds are use of trypsin, use of endonucleases, and use ofexonucleases. Currently, it is recognized in the art that, to fullydecellularize a tissue to produce a scaffold, two or more of theabove-noted ways, and specifically two or more ways from differentgeneral classes (i.e., physical, chemical, enzymatic), should be used.Unfortunately, the methods used must be relatively harsh on the tissueso that complete removal of cellular material can be achieved. The harshtreatments invariable degrade the resulting scaffold, destroyingvasculature and neural structures.

The most successful scaffolds used in both pre-clinical animal studiesand in human clinical applications are biological (natural) and made bydecellularizing organs of large animals (e.g., pigs). In general,removal of cells from a tissue or an organ for preparation of a scaffoldshould leave the complex mixture of structural and functional proteinsthat constitute the extracellular matrix (ECM). The tissues from whichthe ECM is harvested, the species of origin, the decellularizationmethods and the methods of terminal sterilization for these biologicscaffolds vary widely. However, as mentioned above, thedecellularization methods are relatively harsh and result in significantdestruction or degradation of the extracellular scaffold. Once thescaffold is prepared, human cells are seeded so they can proliferate,migrate, and differentiate into the specific tissue. The intent of mostdecellularization processes is to minimize the disruption to theunderlying scaffold and thus retain native mechanical properties andbiologic properties of the tissue. However, to date this intent has notbeen achieved. Snap freezing has been used frequently fordecellularization of tendinous, ligamentous, and nerve tissue. Byrapidly freezing a tissue, intracellular ice crystals form that disruptcellular membranes and cause cell lysis. However, the rate oftemperature change must be carefully controlled to prevent the iceformation from disrupting the ECM as well. While freezing can be aneffective method of cell lysis, it must be followed by processes toremove the cellular material from the tissue.

Cells can be lysed by applying direct pressure to tissue, but thismethod is only effective for tissues or organs that are notcharacterized by densely organized ECM (e.g., liver, lung). Mechanicalforce has also been used to delaminate layers of tissue from organs thatare characterized by natural planes of dissection, such as the smallintestine and the urinary bladder. These methods are effective, andcause minimal disruption to the three-dimensional architecture of theECM within these tissues. Furthermore, mechanical agitation andsonication have been utilized simultaneously with chemical treatment toassist in cell lysis and removal of cellular debris. Mechanicalagitation can be applied by using a magnetic stir plate, an orbitalshaker, or a low profile roller. There have been no studies performed todetermine the optimal magnitude or frequency of sonication fordisruption of cells, but a standard ultrasonic cleaner appears to beeffective. As noted above, currently used physical treatments aregenerally insufficient to achieve complete decellularization, and mustbe combined with a secondary treatment, typically a chemical treatment.Enzymatic treatments, such as trypsin, and chemical treatment, such asionic solutions and detergents, disrupt cell membranes and the bondsresponsible for intercellular and extracellular connections. Therefore,they are often used as a second step in decellularization, after grossdisruption by mechanical means.

It is also recognized in the art that any processing step currentlyknown that is used to remove cells will alter the nativethree-dimensional architecture of the ECM. This is an undesirableside-effect of the treatment, and attempts have been made to minimizethe amount of disruption of the ECM.

SUMMARY OF THE INVENTION

The present invention provides an advancement over tissue engineeringtechniques known in the art. Specifically, the present inventionprovides a method of making engineered tissue scaffolds usingirreversible electroporation (IRE) to decellularize natural tissue. Useof IRE to decellularize tissue provides a controlled, precise way todestroy cells of a tissue or organ, while leaving the underlying ECM,including vascularization, neural tubes, and other gross morphologicalfeatures of the original tissue intact. The decellularized scaffolds arethen suitable for seeding with cells of the appropriate organism. Wherethe process is performed in vitro, the seeded tissue is suitable forimplantation into the organism as replacement tissue. In addition tomethods of producing scaffolds, the invention also provides thedecellularized scaffolds themselves, as well as methods of fabricationof engineered tissues and organs built from such scaffolds. Furthermore,the invention provides for use of the engineered scaffolds and theengineered tissues and organs built from such scaffolds.

Non-thermal IRE is a method to kill undesirable cells using electricfields in tissue while preserving the ECM, blood vessels, and nerves.Certain electrical fields, when applied across a cell, have the abilityto permeabilize the cell membrane through a process that has come to becalled “electroporation”. When electrical fields permeabilize the cellmembrane temporarily, after which the cells survive, the process isknown as “reversible electroporation”. Reversible electroporation hasbecome an important tool in biotechnology and medicine. Other electricalfields can cause the cell membrane to become permeabilized, after whichthe cells die. This deadly process is known as “irreversibleelectroporation”. Non-thermal irreversible electroporation is a new,minimally invasive surgical technique to ablate undesirable tissue, forexample, tumor tissue. The technique is easy to apply, can be monitoredand controlled, is not affected by local blood flow, and does notrequire the use of adjuvant drugs. The minimally invasive procedureinvolves placing needle-like electrodes into or around the targeted areato deliver a series of short and intense electric pulses that inducestructural changes in the cell membranes that promote cell death. Thevoltages are applied in order to electroporate tissue without inducingsignificant joule heating that would significantly damage major bloodvessels and the ECM. For a specific tissue type and set of pulseconditions, the primary parameter determining the volume irreversiblyelectroporated is the electric field distribution within the tissue.Recent IRE animal experiments have verified the many beneficial effectsresulting from this special mode of non-thermal cell ablation, such aspreservation of major structures including the extracellular matrix,major blood vessels, and myelin sheaths, no scar formation, as well asits promotion of a beneficial immune response.

However, the usefulness of IRE in generating tissue scaffolds for tissueengineering has not been recognized. The present invention, for thefirst time, discloses implementation of non-thermal IRE in the widelydivergent field of tissue engineering. Use of non-thermal IRE inpreparing tissue scaffolds not only provides a novel means for achievingthat goal, but addresses long felt needs in the tissue engineeringfield. In various embodiments, the needs that are addressed are:preparation of tissue scaffolds with the underlying matrix essentiallyintact; preparation of tissue scaffolds having the ability to providecirculation, and preferably microcirculation; preparation of tissuescaffolds having the ability to provide spaces for neural infiltration;and preparation of relatively thick (e.g., greater than 100 μm inthickness) engineered tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe invention, and together with the written description, serve toexplain certain principles of the invention.

FIGS. 1A-1C show magnetic resonance imaging (MRI) images of tissue afternon-thermal IRE on canine tissue. The images show that non-thermal IREdecellularization zones were sharply demarcated T1 iso- to hypo-intense,T2 hyperintense and mild and peripherally contrast enhancing followingintravenous administration of gadolinium, consistent with fluidaccumulation within decellularization sites and a focal disruption ofthe blood-brain-barrier. FIG. 1A shows an MRI before IRE, T2 weighted;FIG. 1B shows superficial non-thermal IRE decellularization site, T2weighted; and FIG. 1C shows post-contrast T1 weighted; the dog's rightis conventionally projected on the left.

FIG. 2 shows an ultrasound image of tissue 24 hour post-IRE treatment.The IRE decelluarization zone is clearly visible as a well demarcated,hypoechoic circular lesion with a hyperechoic rim.

FIG. 3 shows photographs of fixed brain sections to show position andcharacter of decellularized volume.

FIGS. 4A and 4B depict images of brain tissue after non-thermal IREtreatment. FIG. 4A shows a sharp delineation of brain tissue showing theregions of normal and necrotic canine brain tissue after IRE. FIG. 4Bshows IRE treated brain tissue showing sparing of major blood vessels.

FIG. 5 shows a three-dimensional MRI source reconstruction of asuperficial lesion site.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to various exemplary embodiments ofthe invention. It is to be understood that the following discussion ofexemplary embodiments is not intended as a limitation on the invention,as broadly disclosed above. Rather, the following discussion is providedto give the reader a more detailed understanding of certain aspects andfeatures of the invention.

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Further, where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lowerlimit, unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Eachsmaller range between any stated value or intervening value in a statedrange and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range,and each range where either, neither, or both limits are included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a pulse” includes aplurality of such pulses and reference to “the sample” includesreference to one or more samples and equivalents thereof known to thoseskilled in the art, and so forth.

Tissue engineering of tissue and organ replacements generally involvesin vitro seeding and attachment of human cells onto a scaffold. To date,the most successful scaffolds for tissue engineering have been naturaland made by chemically and/or mechanically decellularizing organs oflarge animals (e.g., pigs). Such techniques have been successful inmaking scaffolds to build thin organs, such as bladders, which have beensuccessfully implanted in humans. Nevertheless, the field of tissueengineering is currently limited to organs that are less than about 1 mmthick because the process to decellularize the scaffold destroys vitalblood vessels (as well as nerves and other architecture.

The present invention provides decellularize scaffolds, which arecreated at least in part using non-thermal irreversible electroporation(IRE). IRE is a method to kill undesirable cells using electric fields,which is known in the field of medical devices and tumor treatment. Theprocedure involves delivering a series of low energy (intense but short)electric pulses to the targeted tissue. These pulses irrecoverablydestabilize the cell membranes of the targeted tissue, thereby killingthe cells affected by the electrical field. The treatment isnon-thermal, essentially only affects the cell membranes of the targetedtissue, and does not affect the nerves or blood vessels within thetreated tissue. The organ may need to be perfused during the procedure,which is a routine technique in the medical arts.

In a first aspect, the invention provides a method of making adecellularized tissue scaffold. In general, the method comprisestreating, in vitro or in vivo, a tissue comprising cells and anunderlying scaffold with an electrical field of sufficient power andduration to kill cells of the tissue, but not to disrupt to asignificant extent the underlying scaffold. The method is suitable forproducing a tissue scaffold for use in tissue engineering. Although thesource of the tissue is not limited, in exemplary embodiments, thetissue is from a relatively large animal or an animal recognized ashaving a similar anatomy (with regard to the tissue of interest) as ahuman, such as a pig, a cow, a horse, a monkey, or an ape. Inembodiments, the source of the tissue is human, use of which can reducethe possibility of rejection of engineered tissues based on thescaffold. In preferred embodiments, the method leaves intact vascularstructures of the tissue, such as capillaries. In embodiments, themethod leaves intact neural tubes present in the tissue before treatmentwith the electrical field. As used herein, the term “intact” refers to astate of being whereby an element is capable of performing its originalfunction to a substantial extent. Thus, for example, an intact capillaryis a capillary that is capable of carrying blood and an intact neuraltube is a tube that can accommodate a neuron. In embodiments, cells ofthe vascular system and neural system remain intact. In suchembodiments, the cells can remain as part of the scaffold and engineeredtissue, or may be removed by suitable treatment techniques or by cellsthat are seeded onto a scaffold or by cells of a body that receives theengineered tissue.

According to the method, a tissue is exposed to an electrical field thatis adequate in time and power to cause killing of cells of the tissue,but not adequate to significantly destroy the scaffolding upon andwithin which the cells exist. Furthermore, the electrical field does notcause irreversible tissue damage as a result of heating of the tissue.Various ways of providing such an electrical field are possible. Generalparameters follow; however, those of skill in the art are fully capableof devising alternative combinations to achieve the same end resultwithout undue experimentation. In typical embodiments, one or moreelectrical pulses are applied to the tissue to cause cell membranedisruption as a result of the electricity and not substantially as aresult of heat. Where two or more pulses are used, the pulses areseparated by a period of time that allows, among other things, thetissue to cool so that thermal damage does not occur to a significantextent. For example, one or more electrical pulses can be applied to thetissue of interest for a duration in a range of from about 5microseconds (μs) to about 62 seconds. For convenience, a short periodof treatment might be desired. As such, in preferred embodiments,electrical pulses are applied for a period of about 1-10000 μs. Further,although there is no limit on the number of pulses to be delivered tothe tissues, in preferred embodiments, from about 1 to about 100 pulsesare applied to the tissue. For example, in an exemplary embodiment,about 10-1000 pulses of about 100 μs each in duration are applied to thetissue to cause cellular ablation.

There are several parameters that can be monitored and adjusted in usingnon-thermal IRE for preparation of tissue scaffolds. One such parameteris voltage gradient. In some embodiments, the pulses produce a voltagegradient in a range of from about 10 volt/cm to about 10,000 volt/cm.Voltage gradient (electric field) is a function of the distance betweenelectrodes and electrode geometry, which will vary depending on the sizeof the tissue sample, tissue properties, and other factors. In someembodiments, two electrodes are used, and they are placed about 5 mm to10 cm apart. Typical electrode diameters range from 0.25-1.5 mm andtypically 2 or 4 electrodes are used. In embodiments, one bipolarelectrode is used. Also, the “electrode” can have parts of it insulating(including using a non-conductive sheath) and parts of it conductive(e.g., at the tip) to ensure proper application of the electricalcurrent and to minimize production of excessive heat in parts of thetissue.

Appropriate electrical fields and durations of exposure are those thathave been reported in the literature as being suitable for medicaltreatment of tissues for tumor ablation. Exemplary exposure parametersinclude: ninety 90 microsecond (μs) pulses at 1.5 kV/cm at a frequencyof 1 Hz; eighty 100 μs pulses at 2.5 kV/cm at a frequency of 1 Hz; one20 millisecond pulse at 400V/cm; ten 100 μs pulses at 3800 V/cm at afrequency of 10 pulses per second; ninety 100 μs pulses ranging from1000 to 1667 V/cm at a frequency of about 1 Hz; and eighty pulses of 100μs ranging from 1000 to 3000 V/cm at about 1 Hz. In general, thefrequency of pulsing can be as low as twice the pulse width and can bequite a bit farther apart. Any suitable frequency that allows forelectroporation without significant thermal damage to the tissue isacceptable. Furthermore, electrical current can be supplied as either DCor AC.

The shape and size of the electrodes are not critical to practice of theinvention. Those of skill in the art may choose any shape and size thatis suitable for transmitting the desired electrical field into thetissue. For example, the electrodes may be circular in shape, ovoid,square, rectangular, diamond-shaped, hexagonal, octagonal, etc.Likewise, the surface area of the electrodes is not critical to practiceof the invention. Thus, for example, the surface area may be about 0.5square centimeter, about 1 square centimeter, or greater.

Exposing the tissue to the electrical field generates heat. To ensurethat tissue damage due to heat is avoided, the amount of energytransmitted to the tissue is set below a threshold level per unit time.Time and temperature parameters are known in the art for IRE, and anysuitable combination may be used. For example, the temperature of thetissue can be monitored during treatment for ablation, and theelectrical pulses adjusted to maintain the temperature at 100° C. orless, such as 60° C. or less. Preferably, the temperature is maintainedat 50° C. or less.

In some embodiments, the method includes adjusting the applied voltage,length of the pulses, and/or number of pulses to obtain irreversibleelectroporation averaged over the biological cells of the tissue,thereby achieving irreversible electroporation of the biological cellsin the tissue at a level that minimizes damage to non-target tissue.Likewise, in some embodiments, the duration of the applied voltage isadjusted in accordance with the current-to-voltage ratio to achieveirreversible electroporation of identified tissue cells, whereby cellmembranes are disrupted in a manner resulting in cell death. Additionalexemplary parameters are disclosed below.

The present invention thus comprises a method for the creation ofscaffolds, involving the placement of electrodes into or near thevicinity of the original tissue with the application of electricalpulses causing irreversible electroporation of the cells throughout theentire treated region. It is to be noted that placement of theelectrodes defines the treated region; thus, the treated region may beonly a portion of an entire tissue or organ that is used as the startingmaterial. The electric pulses irreversibly permeate the membranes oftreated cells, thereby invoking cell death. The length of time of theelectrical pulses, the voltage applied, and the resulting membranepermeability are all controlled within defined ranges. Application ofelectric pulses results in cell death, but preserves some or all of thevascular and neural structures, preferably including those involved inmicrocirculation. Thus, in some embodiments, microcirculation structuresmay be partially or totally damaged, but larger structures maintained.

For in vitro practice of this aspect of the invention, secondarytechniques for removing cellular material can be used. For example, anyof the known physical, chemical, or enzymatic techniques can be used toremove cellular debris from the irreversibly permeabilized cells.Likewise, the treated tissue can be attached to an artificial perfusionpump, which can pump a liquid composition (e.g., a detergent-containingaqueous composition) through the treated tissue, resulting in removal ofcell debris from the scaffold. Importantly, such secondary treatments,where applied, can be applied under relatively gentle conditions, whichallow for removal of cellular debris but also retention of thescaffolding structure (including vascular and neural structures). Theuse of non-thermal IRE allows for such gentle procedures, and improvesthe scaffold that is ultimately produced, as compared to procedures notrelying on non-thermal IRE.

For in vivo practice of the method, the debris remaining from theirreversibly permeabilized cells may be left in situ and may be removedby natural processes, such as the body's own circulation and immunesystem.

The amount of tissue ablation achievable through the use of irreversibleelectroporation without inducing thermal damage is considerable, asdisclosed and described herein.

The concept of irreversible electroporation to decellularize tissues isdifferent from other forms decellularization used in the art.Irreversible electroporation is different from chemical and physicalmethods or cell lysis via osmotic imbalance because it uses electricityto kill the cells. Irreversible electroporation is a more benign methodbecause it destroys only the cell membrane of cells in the targetedtissue and does no damage to the underlying ECM. Chemical and physicalmethods can damage vital structures, such as the ECM, blood vessels, andnerves. In contrast, IRE of the type described here, solely useselectrical pulses to serve as the active means for inducing cell deathby a specific means, i.e., by fatally disrupting the cell membrane.

Irreversible electroporation may be used for the decellularizing tissuein a minimally invasive procedure that does not or does notsubstantially affect the ECM. Its non-selective mode ofdecellularization is acceptable in the field of tissue engineering andprovides results that in some ways are comparable to sonication,inducing an osmotic imbalance, freezing, or chemical decellularization.

One exemplary embodiment of the invention includes a method wherebycells of tissue are irreversibly electroporated by applying pulses ofvery precisely determined length and voltage. This may be done whilemeasuring and/or observing changes in electrical impedance in real timeand noting decreases at the onset of electroporation and adjusting thecurrent in real time to obtain irreversible cellular damage withoutthermal damage. The method thus may include use of a computing deviceand sensors to monitor the effects of the electrical treatment. Inembodiments where voltage is applied, the monitoring of the impedanceaffords the user knowledge of the presence or absence of pores. Thismeasurement shows the progress of the pore formation and indicateswhether irreversible pore formation, leading to cell death, hasoccurred.

Yet another embodiment includes a method whereby the onset and extent ofelectroporation of cells in tissue can be correlated to changes in theelectrical impedance (which term is used herein to mean the voltage overcurrent) of the tissue. At a given point, the electroporation becomesirreversible. A decrease in the resistivity of a group of biologicalcells occurs when membranes of the cells become permeable due to poreformation. By monitoring the impedance of the biological cells in atissue, one can detect the average point in time in which pore formationof the cells occurs, as well as the relative degree of cell membranepermeability due to the pore formation. By gradually increasing voltageand testing cells in a given tissue, one can determine a point whereirreversible electroporation occurs. This information can then be usedto establish that, on average, the cells of the tissue have, in fact,undergone irreversible electroporation. This information can also beused to control the electroporation process by governing the selectionof the voltage magnitude. Other imaging techniques can be employed tomonitor how much area has been treated (e.g., ultrasound, MRI).

The invention provides the simultaneous irreversible electroporation ofmultitudes of cells providing a direct indication of the actualoccurrence of electroporation and an indication of the degree ofelectroporation averaged over the multitude. The discovery is likewiseuseful in the irreversible electroporation of biological tissue (massesof biological cells with contiguous membranes) for the same reasons. Thebenefits of this process include a high level of control over thebeginning point of irreversible electroporation.

One feature of embodiments of the invention is that the magnitude ofelectrical current during electroporation of the tissue becomesdependent on the degree of electroporation so that current and pulselength are adjusted within a range predetermined to obtain irreversibleelectroporation of targeted cells of the tissue while minimizingcellular damage to surrounding cells and tissue. Yet another feature ofembodiments of the invention is that pulse length and current areprecisely adjusted within ranges to provide more than mere intracellularelectro-manipulation which results in cell death and less than thatwhich would cause thermal damages to the surrounding tissues. Anotherfeature of embodiments is that measuring current (in real time) througha circuit gives a measurement of the average overall degree ofelectroporation that the cells in the tissue achieve.

Yet other features of embodiments include: the precise electricalresistance of the tissue can be calculated from cross-time voltagemeasurement with probe electrodes and cross-current measurement with thecircuit attached to electroporation electrodes; the precise electricalresistance of the tissue is calculated from cross-time voltagemeasurement with probe electrodes and cross-current measurement with thecircuit attached to electroporation electrodes; and electricalmeasurements of the tissue can be used to map the electroporationdistribution of the tissue. It is noted that, in irreversibleelectroporation it is possible and perhaps even preferred to perform thecurrent or EIT measurements a substantial time (several minutes or more)after the electroporation to verify that it is indeed irreversible.

In embodiments of the method, it is preferred to remove cellular debrisfrom the decellularized scaffolding after primary cell destruction withnon-thermal IRE. In such embodiments, any known technique for doing somay be used, including any of the known physical, chemical, and/orenzymatic methods. In one exemplary embodiment, removal of cellularmaterial is accomplished, at least in part, through perfusion of thetissue scaffolding with an appropriate agent (e.g., water, pH-adjustedwater, an aqueous solution of one or more chelating agents, etc.), usinggeneral diffusion, transmittal via remaining intact vasculature, or amixture of the two.

For in vitro methods, it is preferred that the scaffold be sterilized,especially where the scaffold is to be used to prepare engineeredtissues and organs for implantation into a host. Sterilization and/orremoval of debris after decellularization is usually conducted forscaffolds that will be used as implants to reduce the risk of patientrejection (for example, due to DNA fragments). When a scaffold requiressome type of sterilization, methods published in the literature forsterilization of scaffolds can be employed.

For in vitro methods, the method of making a decellularized tissuescaffold results in a decellularized tissue scaffold that is isolatedfrom its natural environment. For in vivo methods, the method of makinga decellularized tissue scaffold results in a tissue scaffold that isdevoid of normal cellular material. Thus, in an aspect of the invention,an engineered tissue scaffold is provided. The engineered tissuescaffold comprises a natural scaffold that is removed from its naturalenvironment and/or from which cellular material has been removed. Theengineered tissue scaffold of the invention contains at least some,preferably most, and more preferably substantially all or all, of thevascular structures (i.e., arteries, veins, capillaries) present in thetissue in its natural state. In embodiments, the tissue scaffoldcomprises at least some, preferably most, and more preferablysubstantially all or all of the neural structures present in the tissuein its natural state. In embodiments, the scaffold further comprises thecells that constitute these vascular structures and/or these neuralstructures. Thus, preferably, the engineered tissue scaffold contains areduced number of the cells naturally populating the scaffold. Inpreferred embodiments, a majority of the original cells, more preferablysubstantially all of the original cells, and most preferably all of theoriginal cells, are absent from the engineered scaffold. In embodiments,the remaining cells are cells that comprise vascular or neuralstructures. Furthermore, in preferred embodiments, some, most, or all ofthe cellular debris from the original cells is absent from theengineered scaffold. Likewise, in embodiments, the tissue scaffoldcontains some or all of the neurons originally present in the tissue.However, in embodiments, the neurons are destroyed but the neural tubesin which the neurons existed remain intact.

In some embodiments, the engineered scaffold comprises cell debris fromcells originally (i.e., naturally) populating the scaffold. As discussedabove, in such embodiments, the cell debris can be removed using knownmeans. Alternatively, some or all of the cell debris may be left in andon the scaffold. In embodiments where cell debris is left on thescaffold, it can be later removed by the action of new cells seeded ontothe scaffold and/or during the process of seeding, infiltration, andgrowth of new cells. For example, where new cells are seeded onto ascaffold comprising cell debris, the action of the new cellsinfiltrating and growing, alone or in combination with a perfusion meansfor feeding and supporting the new cells, can result in removal of thecell debris.

The present invention provides, for the first time, engineered tissuescaffolds that comprise vascular structures that can function inproviding nutrients and gases to cells growing on and in the scaffolds.The use of non-thermal IRE to create the engineered scaffolds permitsretention of these important structures, and thus provides for improvedscaffolds for use in medical, veterinary, and research activities. Theinvention thus provides engineered scaffolds capable of havingrelatively large dimensions. That is, because re-seeded cells growingwithin the inventive scaffolds need not be close (i.e., within 1 mm) toan external surface in order to obtain nutrients and gas, the engineeredscaffolds may be thicker than scaffolds previously known in the art.Engineered scaffolds may have thicknesses of any desirable range, theonly limitation being the ability to generate the appropriate electricalfield to cause decellularization. However, such a limitation is not asignificant physical constraint, as placement of electrodes to effectIRE is easily adjusted and manipulated according to the desires of thepractitioners of the invention.

Engineered scaffolds of the invention can have thicknesses that approachor mimic the thicknesses of the tissues and organs from which they arederived. Exemplary thicknesses range from relatively thin (i.e., 1 mm orless) to moderately thick (i.e., about 5 mm to 1 cm) to relatively thick(i.e., 5 cm or more).

The disclosure, above, has focused on engineered tissue scaffolds andmethods of making them. The invention also encompasses engineeredtissues and methods of making them. In general, the methods of makingengineered tissues comprises: seeding an engineered scaffoldingaccording to the invention with a cell of interest, and exposing theseeded scaffold to conditions whereby the seeded cells can infiltratethe scaffold matrix and grow. Seeding of the scaffold can be by anytechnique known in the art. Likewise, exposing the seeded scaffold toconditions whereby the cells can infiltrate the scaffold and grow can beany technique known in the art for achieving the result. For example, itcan comprise incubating the seeded scaffold in vitro in a commercialincubator at about 37° C. in commercial growth media, which can besupplemented with appropriate growth factors, antibiotics, etc., ifdesired. Those of skill in the art are fully capable of selectingappropriate seeding and proliferation techniques and parameters withouta detailed description herein. In other words, with respect to seedingand growth of cells, the scaffolds of the present invention generallybehave in a similar manner to other natural scaffolds known in the art.Although the present scaffolds have beneficial properties not possessedby other scaffolds, these properties do not significantly affect seedingand growth of cells.

Engineered tissues have been developed as replacements for injured,diseased, or otherwise defective tissues. An important goal in the fieldof tissue engineering is to develop tissues for medical/therapeutic usein human health. In view of the difficulty and ethical issuessurrounding use of human tissues as a source of scaffolds, tissues fromlarge animals are typically used for the source material for naturalscaffolds. The xenotypic scaffolds are then seeded with human cells foruse in vivo in humans. While the presently disclosed engineered tissuesare not limited to human tissues based on animal scaffolds, it isenvisioned that a primary configuration of the engineered tissues willhave that make-up. Thus, in embodiments, the engineered tissues of theinvention are tissues comprising human cells on and within a scaffoldderived from an animal tissue other than human tissue.

For certain in vivo uses, animal tissue is subjected in vivo tonon-thermal IRE, and the treated tissue cleared of cell debris by thehost animal's body. Thus, in certain in vivo embodiments, no secondarycell debris removal step is required, as the host animal's body itselfis capable of such removal (this concept applies to in vivo creation ofscaffolds in humans as well). The treated tissue is then seeded in vivo,for example with human cells, and the seeded cells allowed to infiltratethe scaffold and grow. Upon suitable infiltration and growth, theregenerated tissue is removed, preferably cleaned of possiblecontaminating host cells, and implanted into a recipient animal, forexample a human. In such a situation, it is preferred that the hostanimal is one that has an impaired immune system that is incapable orpoorly capable of recognizing the seeded cells as foreign cells. Forexample, a genetically modified animal having an impaired immune systemcan be used as the host animal. Alternatively, for example, the hostanimal can be given immune-suppressing agents to reduce or eliminate theanimal's immune response to the seeded cells.

It is important to note at this point that the recipient can be anyanimal. It thus can be a human or another animal, such as a companionanimal (i.e., a pet, such as a dog or cat), a farm animal (e.g., abovine, porcine, ovine), or a sporting animal (e.g., a horse). Theinvention thus has applicability to both human and veterinarian healthcare and research fields.

Whether in vivo or in vitro, the choice of cells to be seeded will beleft to the practitioner. Many cell types can be obtained, and those ofskill in the tissue engineering field can easily determine which type ofcell to use for seeding of tissues. For example, one may elect to usefibroblasts, chondrocytes, or hepatocytes. In embodiments, embryonic oradult stem cells, such as mesenchymal stem cells, are used to seed thescaffolds. The source of seeded cells is not particularly limited. Thus,the seeded cells may be autologous, syngenic or isogenic, allogenic, orxenogenic. However, because a feature of the present invention is theproduction of scaffolds and tissues that have reduced immunogenicity (ascompared to scaffolds and tissues currently known in the art), it ispreferred that the seeded cells be autologous (with relation to therecipient of the engineered tissue). In certain embodiments, it ispreferred that the seeded cells be stem cells or other cells that areable to differentiate into the proper cell type for the tissue ofinterest.

Alternatively or additionally, the in vivo method of creating a scaffoldand the in vivo method of creating an engineered tissue can includetreating tissue near the non-thermal IRE treated cells with reversibleelectroporation. As part of the reversible electroporation, one or moregenetic elements, proteins, or other substances (e.g., drugs) may beinserted into the treated cells. The genetic elements can include codingregions or other information that, when expressed, reduces interactionof the cells with the seeded cells, or otherwise producesanti-inflammatory or other anti-immunity substances. Short-termexpression of such genetic elements can enhance the ability to growengineered tissues in vivo without damage or rejection. Proteins andother substances can have an effect directly, either within thereversibly electroporated cells or as products released from the cellsafter electroporation.

Certain embodiments of the invention relate to use of human scaffoldsfor use in preparation of engineered human tissues. As with otherengineered tissues, such engineered tissues can be created in vitro, invivo, or partially in vitro and partially in vivo. For example, tissuedonors may have part or all of a tissue subjected to non-thermal IRE toproduce a scaffold for tissue engineering for implantation of arecipient's cells, then growth of those cells. Upon infiltration andgrowth of the implanted cells, the tissue can be removed and implantedinto the recipient in need of it. Of course, due to ethical concerns,the donor tissue should be tissue that is not critical for the life andhealth of the donor. For example, the tissue can be a portion of aliver. The engineered tissue, upon removal from the host and implantedin the recipient, can regenerate an entire functional liver, while theremaining portion of the host's liver can regenerate the portionremoved.

Up to this point, the invention has been described in terms ofengineered tissue scaffolds, engineered tissues, and methods of makingthem. It is important to note that the invention includes engineeredorgans and methods of making them as well. It is generally regarded thatorgans are defined portions of a multicellular animal that perform adiscrete function or set of functions for the animal. It is furthergenerally regarded that organs are made from tissues, and can be madefrom multiple types of tissues. Because the present invention isgenerally applicable to both tissues and organs, and the distinctionbetween the two is not critical for understanding or practice of theinvention, the terms “tissue” and “organ” are used hereininterchangeably and with no intent to differentiate between the two.

Among the many concepts encompassed by the present invention, mentionmay be made of several exemplary concepts relating to engineeredtissues. For example, in creating engineered organs, the initial organcan be completely removed of cells using irreversible electroporationprior to reseeding (this is especially relevant for organs having atleast one dimension that is less than 1 mm); the organ can beirreversibly electroporated in sections and reseeded to allow the humancells to infiltrate small sections at a time; the organ can beirreversibly electroporated in incremental slices introducing the cellsin stages, so that no human viable cells are in contact with the viableanimal cells they are replacing; the organ can be irreversiblyelectroporated entirely or in sections and the human cells can beinjected into targeted locations in the organ; the entire organ can beirreversibly electroporated to kill the animal cells, then human cellscan be replanted on top of the organ to infiltrate the scaffold andreplace the other cells (as the animal cells die, the human cells willfill in and substitute, thereby creating a new organ.)

Having provided isolated engineered tissues and organs, it is possibleto provide methods of using them. The invention contemplates use of theengineered tissues in both in vitro and in vivo settings. Thus, theinvention provides for use of the engineered tissues for researchpurposes and for therapeutic or medical/veterinary purposes. In researchsettings, an enormous number of practical applications exist for thetechnology. One example of such applications is use of the engineeredtissues in an ex vivo cancer model, such as one to test theeffectiveness of various ablation techniques (including, for example,radiation treatment, chemotherapy treatment, or a combination) in a lab,thus avoiding use of ill patients to optimize a treatment method. Forexample, one can attach a recently removed liver (e.g., pig liver) to abioreactor or scaffold and treat the liver to ablate tissue. Anotherexample of an in vivo use is for tissue engineering.

The engineered tissues of the present invention have use in vivo. Amongthe various uses, mention can be made of methods of in vivo treatment ofsubjects (used interchangeably herein with “patients”, and meant toencompass both human and animals). In general for certain embodiments,methods of treating subjects comprise implanting an engineered tissueaccording to the invention into or on the surface of a subject, whereimplanting of the tissue results in a detectable change in the subject.The detectable change can be any change that can be detected using thenatural senses or using man-made devices. While any type of treatment isenvisioned by the present invention (e.g., therapeutic treatment of adisease or disorder, cosmetic treatment of skin blemishes, etc.), inmany embodiments, the treatment will be therapeutic treatment of adisease, disorder, or other affliction of a subject. As such, adetectable change may be detection of a change, preferably animprovement, in at least one clinical symptom of a disease or disorderaffecting the subject. Exemplary in vivo therapeutic methods includeregeneration of organs after treatment for a tumor, preparation of asurgical site for implantation of a medical device, skin grafting, andreplacement of part or all of a tissue or organ, such as one damaged ordestroyed by a disease or disorder (e.g., the liver). Exemplary organsor tissues include: heart, lung, liver, kidney, urinary bladder, brain,ear, eye, or skin. In view of the fact that a subject may be a human oranimal, the present invention has both medical and veterinaryapplications.

For example, the method of treating may be a method of regenerating adiseased or dysfunctional tissue in a subject. The method can compriseexposing a tissue to non-thermal IRE to kill cells of the treated tissueand create a tissue scaffold. The method can further comprise seedingthe tissue scaffold with cells from outside of the subject, and allowingthe seeded cells to proliferate in and on the tissue scaffold.Proliferation produces a regenerated tissue that contains healthy andfunctional cells. Such a method does not require removal of the tissuescaffold from the subject. Rather, the scaffold is created from theoriginal tissue, then is re-seeded with healthy, functional cells. Theentire process of scaffold creation, engineered tissue creation, andtreatment of the subject is performed in vivo, with the possibleexception of expansion of the cells to be seeded, which can beperformed, if desired, in vitro.

In yet another exemplary embodiment, a tissue scaffold is created usingnon-thermal IRE to ablate a tissue in a donor animal. The treated tissueis allowed to remain in the donor's body to allow the body to clearcellular debris from the tissue scaffold. After an adequate amount oftime, the treated tissue is removed from the donor's body and implantedinto the recipient's body. The transplanted scaffold is not reseededwith external cells. Rather, the scaffold is naturally reseeded by therecipient's body to produce a functional tissue.

The present invention eliminates some of the major problems currentlyencountered with transplants. The methods described herein reduce therisk of rejection (as compared to traditional organ transplants) becausethe only cells remaining from the donor organ, if any, are cellsinvolved in forming blood vessels and nerves. Yet at the same time,vascular and neural structures are maintained. As a result, the presentinvention provides a relatively rapid, effective, and straightforwardway to produce engineered tissues having substantially natural structureand function, and having reduced immunogenicity. As such, the engineeredtissues of the present invention are highly suitable for therapeutic andcosmetic use, while having a relatively low probability of rejection. Inembodiments where human organs are used as the source of the scaffold(e.g., from live organ donors or cadavers), the risk of rejection isvery small.

EXAMPLES

The invention will be further explained by the following Examples, whichare intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way.

As a general background to the Examples, it is noted that the inventorand his colleagues have successfully demonstrated decellularizationusing IRE 1) in vivo and ex vivo, 2) to show that different tissues canbe utilized, 3) to show that the area affected can be predicted usingnumerical modeling, 4) to show how numerical modeling can be used toensure the ECM, blood vessels, and nerves are not thermally damaged, 5)while the organ was attached to a perfusion system, 6) whiledemonstrating preservation of major vasculature and ECM, and 7) withverification through imaging.

Ideally IRE performed ex vivo should be done as the tissue is perfusedin a bioreactor. Perfusion of tissue in a bioreactor has been publishedin the literature, and the parameters disclosed therein can be generallyapplied within the present context. IRE is a special mode for cellablation perfectly suitable to creating scaffolds because it kills thecells in the targeted area while sparing major blood vessels, connectivetissue, nerves, and the surrounding tissue. Typically, mild enzymes orchemicals (non-ionic detergents, zwitterionic detergents, chelatingagents, enzymatic methods) are used to facilitate removal of DNAfragments after decellularization. (It should be noted that for IRE invivo, the removal of cells can be accomplished by the body's naturalsystem.

The following is an “ideal” approach to implementing IRE ex vivo with abioreactor perfusion system:

a) attach freshly excised organ to bioreactor perfusion system tomaintain physiological environment (e.g., 37° C.);

b) perfuse organ with saline;

c) insert electrodes into targeted area;

d) apply IRE pulses;

e) optional: use gentle chemical (e.g., non-ionic detergent) or physicaltechnique to remove cellular content/debris;

f) seed cells into the targeted/treated area;

g) perfuse organ with nutrients/growth media (demonstration of perfusionduring IRE in Edd et al., 2006);

h) maintain bioreactor perfusion system at optimal conditions for cellgrowth (37° C.)

i) optional: repeat steps b-h until entire desired volume of tissue hasbeen treated (e.g., to treat the entire organ).

It is to be noted that the order can be switched in many of these items.

Example 1: IRE on Freshly Excised Mouse Tissue to Create a Scaffold: IREScaffold Test Protocol

A single mouse was sacrificed via CO₂ asphyxiation and the liver wassurgically removed. Two round sections (1 experimental, 1 control) wereremoved from the liver using a 5 mm core borer. A straight edge was thencut into each section to facilitate orientation and imaging. The firstsection was subjected to eighty 100 μs 2500V/cm pulses at 4 Hz. Thesecond section was not treated and left as control. Each section wasthen divided into 5 samples using a scalpel. The outer samples werediscarded, leaving three samples from the experimental section and threesamples from the control section.

One sample from the experimental section and one sample from the controlsection were subjected to sonication for 2 hours at 37° C. One samplefrom the experimental section and one sample from the control sectionwere subjected to agitation via stir bar for 2 hours at 37° C. with arotational rate of 60-300 rpm. One sample from the experimental sectionand one sample from the control section were placed in a water bath for2 hours at 37° C.

Each of the experimental samples was then cut in half. Each section wasthen rinsed twice in DI water. Half of the experimental samples werefixed in formaldehyde for histology. The remaining experimental samplesand all of the control samples were placed in individual 1.5 mLmicro-vials of DI water and flash frozen in liquid nitrogen. The sampleswere freeze dried for 24-48 hours prior to imaging.

Results indicated that the experimental parameters were adequate forcell ablation of the tissue. Furthermore, no thermal damage to tissuewas observed, and ECM, blood vessels, and nerves were preserved. Usingthis protocol and other parameters disclosed herein, various differentIRE protocols can be designed to ensure no thermal damage to ECM, bloodvessels, and nerves. Furthermore, highly customizable fielddistributions can be attained using different electrode geometries.Also, as shown in Edd and Davalos, 2007, tissue heterogeneity can beaccounted for using numerical models.

Example 2: IRE Performance Indicia

To illustrate 1) the possibility to monitor creation of the scaffold inreal-time using imaging techniques, 2) the variety of tissues that canbe used, and 3) how to preserve vasculature, a healthy female purposebred beagle was used. Nine sets of ten pulses were delivered withalternating polarity between the sets to prevent charge build-up on theelectrode surfaces. The maximum voltage-to-distance ratio used was 2000V/cm because the resulting current did not exceed 2 amps. The chargethat was delivered to the brain during the IRE procedure was 22.5 mC,assuming ninety pulses (50 μs pulse durations) that would result from amaximum hypothetical current of 5 amps.

TABLE 1 IRE pulse parameters EXPOSURE GAP VOLTAGE TO PULSE ELECTRODELENGTH DISTANCE VOLTAGE DISTANCE DURATION TYPE [mm] [mm] [V] RATIO[V/cm] PULSES [μs] 1 mm 5 5 500 1000 90 50 Bipolar Standard 7 1600 200090 50

Method: After induction of general anesthesia, a routine parietotemporalcraniectomy defect was created to expose the right temporal lobe of thebrain. Two decelluarization sites were performed: 1) a deep lesionwithin the caudal aspect of the temporal lobe using a monopolarelectrode configuration (6 mm electrode insertion depth perpendicular tothe surface of the target gyrus, with 5 mm interelectrode distance), and2) a superficial lesion in the cranial aspect of the temporal lobe usinga bipolar electrode (inserted 2 cm parallel to the rostrocaudal lengthof the target gyms, and 2 mm below the external surface of the gyms).Intraoperative adverse effects that were encountered included grossmicrohemorrhages around the sharp monopolar electrode needles followinginsertion into the gyms. This hemorrhage was controlled with topicalapplication of hemostatic foam. Subject motion was completelyobliterated prior to ablating the superficial site by escalating thedose of atracurium to 0.4 mg/kg. Grossly visible brain edema and surfaceblanching of the gyrus overlying the bipolar electrode decelluarizationsite was apparent within 2 minutes of completion of IRE at this site.This edema resolved completely following intravenous administration of1.0 g/kg of 20% mannitol. No adverse clinically apparent effectsattributable to the IRE procedure, or significant deterioration inneurologic disability or coma scale scores from baseline evaluationswere observed.

Methods to monitor creation of scaffold: A unique advantage of IRE tocreate scaffolds is its ability to be monitored in real-time usingimaging techniques, such as electrical impedance tomography, MRI, andultrasound. Below, this Example shows MRI examinations performedimmediate post-operatively, which demonstrate that IRE decelluarizationzones were sharply demarcated (FIGS. 1A-C).

As shown in FIGS. 1A-1C, neurosonography performed intraoperatively andat 1 hour and 24 hours post-procedure demonstrated clearly demarcateddecellularization zones and visible needle tracts within the targetedbrain parenchyma. Intraoperatively and immediately postoperatively, thedecellularization zones appeared as hypoechoic foci with needle tractsappearing as distinct hyperechoic regions (FIG. 2).Neurosonographically, at the 24 hour examination the IREdecellularization zone was hypoechoic with a hyperechoic rim (FIG. 2).Compared to the 1 hour post-operative sonogram, the IRE decelluarizationzone appeared slightly larger (1-2 mm increase in maximal, twodimensional diameter). EEG performed in the post-operative periodrevealed focal slowing of the background rhythm over the right temporalregion in association with the decelluarization zones.

Macrolevel and histologic verification of treating cells: The brain wascollected within 2 hours of the time of death and removed from thecranium. Care was taken to inspect soft tissues and areas of closurecreated at the time of surgery. The brain was placed in 10% neutralbuffered formalin solution for a minimum of 48 hours. Then, the brainwas sectioned at 3 mm intervals across the short axis of the brain, inorder to preserve symmetry and to compare lesions. Following grossdissection of fixed tissues, photographs were taken of brain sections inorder to document the position and character of lesions as shown in FIG.3. Readily apparent in gross photographs of the sectioned brain arelesions created either by the physical penetration of brain substancewith electrodes or created by the application of pulse through theelectrodes. There are relatively well-demarcated zones of hemorrhage andmalacia at the sites of pulse delivery.

Microscopic lesions correlated well with macroscale appearance. Areas oftreatment are represented by foci of malacia and dissociation of whiteand grey matter. Small perivascular hemorrhages are present and there issparing of major blood vessels (see FIG. 4B). Notable in multiplesections is a relatively sharp line of demarcation (approximately 20-30μm) between areas of frank malacia and more normal, organized brainsubstance (see FIG. 4A).

Analysis to determine IRE threshold: To determine the electric fieldneeded to irreversibly electroporate tissue, one can correlate thelesion size that was observed in the ultrasound and MRI images with thatin the histopathological analysis to determine the percentage of lesiongrowth. Decellularized site volumes can be determined afteridentification and demarcation of IRE decellularization zones fromsurrounding brain tissue using hand-drawn regions of interest (ROI). Arepresentative source sample image is provided in FIG. 5.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

The invention claimed is:
 1. An in vitro method of making an engineeredtissue scaffold, said method comprising: engineering a tissue scaffoldby treating, in vitro, a tissue comprising cells and a nativeextracellular matrix with an electrical field; applying the electricalfield in a manner and for a sufficient time at a sufficient power tocause irreversible electroporation of the cells; and producing anengineered tissue scaffold as a result of the irreversibleelectroporation.
 2. The method of claim 1, wherein treating comprisesplacing a first electrode and a second electrode in contact with thetissue such that the portion of tissue to be decellularized byirreversible electroporation is positioned between the first and secondelectrodes.
 3. The method of claim 2, wherein more than two electrodesare placed in contact with the tissue.
 4. The method of claim 2, whereintreating with an electrical field comprises applying more than oneelectrical pulse between the first and second electrodes in an amountsufficient to induce irreversible electroporation of cells of thetissue.
 5. The method of claim 1, further comprising removing cellulardebris remaining from the irreversible electroporation.
 6. The method ofclaim 5, wherein the cellular debris is removed using one or morephysical, chemical, or enzymatic techniques.
 7. The method of claim 1,wherein the irreversible electroporation does not cause substantialdestruction of the extracellular matrix due to heat.
 8. The method ofclaim 1, wherein the engineered tissue scaffold comprises a mixture ofstructural and functional proteins.
 9. The method of claim 8, whereinthe engineered tissue scaffold retains the three-dimensionalarchitecture of the native extracellular matrix.
 10. The method of claim8, wherein the engineered tissue scaffold retains the mechanicalproperties of the native extracellular matrix.
 11. The method of claim1, wherein the engineered tissue scaffold has a thickness thatapproaches or mimics the thickness of the tissue before treatment. 12.The method of claim 1, wherein the engineered tissue scaffold has athickness of about 5 mm or more.
 13. The method of claim 1, wherein thetissue is an organ.
 14. The method of claim 13, wherein the organ is aheart, a lung, a liver, a kidney, a urinary bladder, a brain, an ear, aneye, or skin.
 15. The method of claim 1, wherein the tissue furthercomprises one or more vascular structures, and the irreversibleelectroporation does not cause irreversible damage to at least some ofthe one or more vascular structures.
 16. The method of claim 15, whereinat least one of the one or more vascular structures is an artery, vein,or capillary which is left intact in the engineered tissue scaffoldafter the irreversible electroporation.
 17. The method of claim 15,wherein at least one of the one or more vascular structures is leftintact in the engineered tissue scaffold after the irreversibleelectroporation and is capable of conducting microcirculation and/or hasa cross-sectional diameter of 5 to 10 micrometers.
 18. The method ofclaim 15, wherein at least one of the one or more vascular structures isleft intact in the engineered tissue scaffold after the irreversibleelectroporation and comprises a network of arteries, veins, and/orcapillaries.
 19. The method of claim 1, wherein the tissue furthercomprises one or more neural structures, and the irreversibleelectroporation does not cause irreversible damage to at least some ofthe one or more neural structures.
 20. The method of claim 19, whereinat least one of the one or more neural structures is a neural tube whichis left intact in the engineered tissue scaffold after the irreversibleelectroporation.